A DNA ligase is an enzyme that forms phosphodiester bonds -- it can "glue" together two pieces of DNA. The ligase we're using comes from the T4 bacteriophage virus. Why do we need to do a ligation anyway? Remember, we're going to use a Cas9 protein to "cut" your target gene in a yeast cell. We can program the Cas9 to cut a particular place using a "guide RNA." However, does Saccharomyces cerevisiae -- brewers' yeast -- make Cas9 or a guide RNA on its own? No! So we need to give the yeast cell a

more »

... set of instructions for making the Cas9 protein and the guide RNA. We do so by encoding those instructions on a plasmid -- a small circular piece of DNA -- and then putting that plasmid into the yeast cell. Then, the yeast cell "follows" the instructions to make the Cas9 protein and guide RNA. The plasmid we're making has several functional DNA sequences on it -- a diagram is below. The functional sequences on the plasmid are denoted by colored boxes -- you can ignore most of them for now. Note, however, the boxes labelled Cas9 and Guide RNA -- these are the instructions for making Cas9 and the guide RNA. Remember, though, that each group is knocking out a different gene -- so each group needs a different plasmid! The part that needs to be customized for each group is the "target sequence" in the above diagram. Your instructor started with a plasmid similar to the one above, but where the target sequence is instead replaced by gene for a green fluorescent protein (GFP) -- cells that express it turn bright green! They have cut out the GFP, and now you need to replace it with a targeting sequence for your gene, as in the diagram below. One last thing. Where is your targeting sequence coming from? It's the annealed oligonucleotides from last time. You'll mix the plasmid backbone, your annealed oligos, the ligase enzyme, and a ligase buffer (which contains all of the chemicals the ligase needs to operate), and then the ligation will incubate at room temperature while the enzyme does its thing.